Silver(I) N-Heterocyclic Carbene Complexes Derived from Clotrimazole: Antiproliferative Activity and Interaction with an Artificial Membrane-Based Biosensor

Article abstract of DOI:10.1021/acs.organomet.0c00069

With the aim of combining the potential anticancer properties of both clotrimazole, an imidazole based antifungal agent, and silver(I) N-heterocyclic carbenes, 13 novel silver(I) N-heterocyclic carbene complexes derived from clotrimazole were synthesized.

Full text of DOI:10.1021/acs.organomet.0c00069

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Article
Silver(I) N‑Heterocyclic Carbene Complexes Derived from
Clotrimazole: Antiproliferative Activity and Interaction with an
Artificial Membrane-Based Biosensor
Heba A. Mohamed, Samantha Shepherd, Nicola William, Helen A. Blundell, Madhurima Das,
Christopher M. Pask, Benjamin R. M. Lake, Roger M. Phillips,* Andrew Nelson,*
and Charlotte E. Willans*
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ABSTRACT: With the aim of combining the potential anticancer properties of both clotrimazole, an imidazole based antifungal
agent, and silver(I) N-heterocyclic carbenes, 13 novel silver(I) N-heterocyclic carbene complexes derived from clotrimazole were
synthesized. The complexes were fully characterized, and the partition coefficient of each was determined to provide a measure of
hydrophobicity. The antiproliferative properties of the complexes against cancerous and noncancerous cell lines found optimum
cytotoxicity when the complex displays an “intermediate lipophilicity”, which describes a complex that possesses both water-soluble
groups and lipophilic aromatic groups. The silver complexes were screened on a synthetic biomembrane-like device using a chip-
based phospholipid-coated Pt/Hg electrode embedded in a flow cell system. The results are recorded as rapid cyclic voltammograms
(RCVs), which give insight into the interactions of the complexes with a cell membrane. Interestingly the principle of “intermediate
lipophilicity” also applies to the monolayer interaction to which the silver atom significantly implements an irreversibility.
Over the last few decades, N-heterocyclic carbenes (NHCs)
have become ubiquitous ligands in organometallic chemistry,
particularly in the field of catalysis.14−23 In recent years, metal-
NHCs have shown promise in biomedical applications,
including as antimicrobial (silver-NHCs) and as antitumor
(palladium-, copper-, silver-, gold-, ruthenium-, and platinum-
NHCs) agents.12,24−41 The efficacy of a silver-based drug
appears to be linked to its bioavailability,29,42 which is affected
by, for example, its solubility and the presence of biological
ligands and halides. The release rate of silver from a prodrug is
INTRODUCTION
■
The serendipitous discovery of cisplatin as an anticancer agent
by Rosenberg at the end of the 1960s opened up a new area of
research in organometallic chemistry for the treatment of
cancer.1 Despite the success of cisplatin and other platinum-
based drugs, severe side effects and the development of drug
resistance are drawbacks to their clinical applications.2,3
Therefore, research efforts have also examined the potential
of other metals as chemotherapeutic agents. Complexes of
ruthenium,4−6 iron,7 and titanium8 exhibit significant cytotoxic
effects, with some ruthenium complexes having undergone
clinical trials.9 Silver is another metal that has been studied for
its biomedicinal properties, as it is thought to have relatively
low toxicity, and the antimicrobial properties of this metal have
been exploited for centuries.10 In recent years, studies have
shown that silver also has potential in the field of cancer
chemotherapy.11−13
Received: February 3, 2020
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linked to the ancillary ligand, and as NHCs are strong σ
donors, silver-NHCs can have a slow silver release rate. It is
imperative that the ligand also have a low toxicity profile.
Clotrimazole (Figure 1A) is an imidazole-containing
compound and is a highly effective treatment for fungal
clotrimazole-derived imidazolium salts. The complexes were
screened against cancerous cell lines, including the pancreatic
cancer cell lines Panc 10.05 and MIA PaC-2, and the colorectal
carcinoma BE cell line, plus the noncancerous cell line APRE-
19 in order to assess both their cytotoxicity and selectivity. As
clotrimazole inhibits proliferating cells by affecting the
transport of ions across the plasma membrane,45 the silver
complexes and corresponding ligand precursors have been
screened on a biosensing device which measures their
interaction with a phospholipid sensor element, to provide
an indication of their biological membrane activity.47
RESULTS AND DISCUSSION
■
Figure 1. Active antifungal compound clotrimazole (A) and its
metabolites 2-chlorobenzophenone (B) and (2-chlorophenyl)-
diphenylmethanol (C).
Imidazolium salts 1a,c were prepared using microwave
conditions (Scheme 1A) and fully characterized. Crystals
suitable for X-ray crystallography were grown via diffusion of
diethyl ether into a solution of either 1a or 1c in
dichloromethane (see the Supporting Information). The
solid-state structures show the expected imidazolium iodide
salts, with no evidence of hydrogen-bonding or anion−π
interactions between the iodide anion and the imidazolium
ring.48 Such interactions are likely to be precluded by the
presence of the bulky trityl group adjacent to the imidazolium
ring. Interestingly, an anion−π interaction does appear to exist
between the aryl chloride and the imidazolium ring in 1c, with
an aryl-Cl···imidazolium centroid distance of 3.28 Å. In
contrast to 1a,c, we found that imidazolium salt 1e could
instead be prepared more efficiently through the reaction of
the reagents in dichloromethane at reflux (Scheme 1B).
Crystals suitable for X-ray crystallography were grown via
diffusion of diethyl ether into a solution of 1e in dichloro-
methane. The structure shows the expected imidazolium salt;
diseases in plants, humans, and animals.43 It is a readily
available material that is already present in many pharmaceut-
ical treatments, administered both orally and topically for
infections such as dermatophytes and staphylococci. Once
administered, clotrimazole can be metabolized and excreted
from the body (Figure 1B,C).44 Clotrimazole also shows
activity against various tumor cell lines and is thought to be
effective by decreasing the movement of intracellular Ca2+ and
K+ ions.45 Its ability to interact with cell membranes and
penetrate cells can be attributed to its aromaticity.46 As
clotrimazole is an imidazole-containing compound, possessing
negligible toxicity and anticancer properties, it is an ideal
precursor to silver-NHCs for examination as chemotherapeutic
agents. Herein we report the synthesis, characterization, and
bioactivity of a family of silver(I)-NHCs prepared from
Scheme 1. Synthesis of Imidazolium Salts 1a,c,e starting from Clotrimazole (A, B), 2a−e Starting from 2-Chlorotrityl Chloride
a
(C), and 3a−e Starting from Trityl Chloride (D) and Silver-NHC Complexes
a
In the box are shown solution equilibria between 2Ag(NHC)X and [Ag(NHC)2]AgX2 with removal of AgX to form [Ag(NHC)2]X.
B
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Figure 2. Molecular structures of silver-NHC complexes 4e, 5a,b,d, and 6c. Hydrogen atoms and water molecules have been omitted for clarity.
Ellipsoids are shown at 50% probability for 5a,b, 40% for 5d, and 35% for 4e and 6c.
−
however, the anion (labeled I(1)) is substitutionally dis-
ordered, containing a mixture of chloride (29%) and iodide
(71%) (see the Supporting Information). The chloride may
originate from impurities in the dichloromethane solvent;
however, it is likely that this observation is a result of exchange
between the iodide anion and the chloride of the 2-chlorotrityl
group. This is further evidenced through the aryl halide group
(labeled Cl(1)) also being present as a mixture of chloride
(97%) and iodide (3%). To prevent the formation of
imidazolium salts with disordered halides, N-substituted
imidazoles were reacted with 2-chlorotrityl chloride in
acetonitrile or dichloromethane at reflux, to yield imidazolium
compounds 2a−e (Scheme 1C). With the intent of
investigating the effect of the 2-chlorotrityl group within the
ligand on the anticancer activity of the silver complexes, a
second set of imidazolium compounds 3a−e were synthesized
in the same manner, starting with trityl chloride (Scheme 1D).
The compounds were fully characterized, with crystals of
2a,b,d,e and 3e suitable for X-ray diffraction analysis being
grown by diffusion of either diethyl ether or pentane into
either a dichloromethane or methanol solution of the
compounds (see the Supporting Information).
halide), [Ag(NHC)2]X (where X = halide and/or AgX2 ), or a
combination of the two. Solution equilibria between 2Ag-
(NHC)X and [Ag(NHC)2]AgX2 is a common phenomenon
observed in silver-NHCs and is not observed on the NMR
time scale here (Scheme 1).13,29,49−51 Equilibration of these
species is known to occur on very different time scales in
different media.52 As the complexes were filtered through
Celite during workup, some AgX was removed from the bis-
NHCs, resulting in X rather than AgX2 counterions in the
cationic species (X = Cl, I). Silver-NHC complexes were fully
characterized using NMR spectroscopy, high-resolution mass
spectrometry, and elemental analysis, which revealed the ratios
of X− to AgX2 counteranions, and solid-state structures were
−
obtained for complexes 4e, 5a,b,d, and 6c (Figure 2). The
molecular structures reveal that the mono- and bis-NHC
complexes have similar bond distances between the silver and
the carbenic carbon atoms (2.08−2.12 Å) and similar NCN
bond angles between the carbenic carbon and neighboring
nitrogen atoms (103−106°). The geometry around the silver
centers distort slightly from linearity, with Ccarbene−Ag−
C
carbene/X bond angles ranging 171.2(7)° for 6c to
176.42(14) ° for 5a.
Reaction of the imidazolium salts with Ag2O resulted in the
formation of complexes of the type Ag(NHC)X (where X =
As a means to assess the affinity of complexes 4−6 for the
phospholipid component of the cell membrane, the partition
C
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coefficient (log P) of the 13 complexes and cisplatin was
measured. The results are summarized in Figure 3, with log P
than that of cisplatin (4a,e and 5e) were evaluated further by
assessing their antiproliferative activity against pancreatic
carcinoma MIA PaC-2 and colorectal carcinoma BE.
Complexes 4b,d and 6a,c,e were also tested against all four
cell lines to determine whether low selectivity ratios were also
observed in other cell lines, and the results are summarized in
Figure 4 and Table 1. From the MTT assays it can be seen that
the 2-chlorotrityl-bearing silver-NHC complexes 4a−e are
generally more cytotoxic against Panc 10.05 than their
corresponding trityl-bearing complexes 5a−e, indicating that
the presence of an aryl-Cl group plays a role in the activity of
these complexes. This could be due to the fact that the Cl
group increases the propensity of the complexes to interact
with phospholipid and cell membranes.53 Complex 4e is the
most active against Panc 10.05 of all 13 complexes tested and
exhibits an IC50 value which is comparable to that of cisplatin
(p > 0.05). The hydroxyethyl group increases the hydro-
philicity of the complex (lower log P), thus enhancing the
water solubility, which also improves the cytotoxicity.
Changing the counterion from chloride on complex 4c to
iodide in complex 6c improves the cytotoxicity against Panc
10.05 (p < 0.01). However, when iodide complex 6e was tested
against Panc 10.05, it revealed a numerically inferior activity in
comparison to its corresponding chloride-containing analogue
4e (although the difference did not reach statistical
significance; p > 0.05), with complexes 4a (chloride) and 6a
(iodide) exhibiting activities similar to each other (p > 0.05).
In general, iodide-containing complexes such as 6c displays
comparable cytotoxicity against all cell lines tested in
comparison to its chloride analogues 4a (p > 0.05) and 4c
Figure 3. Bar chart to show the log P values for complexes 4−6.
values ranging from 0.26( 0.06) (5e) to more hydrophobic
0.59( 0.07) (6c). Cisplatin, with a log P value of
−1.35( 0.26), is more hydrophilic than any of the silver-
NHC complexes tested. Three trends relating to hydro-
phobicity can be observed from Figure 3. (i) Complexes 4a−e,
with a Cl substituent on the triphenyl group, appear to be
more hydrophobic than the corresponding complexes 5a−e,
where the Cl is replaced with a H. (ii) The benzyl-containing
complexes 4d and 5d are the most hydrophobic in their series
followed by butyl-, allyl-, methyl-, and hydroxyethyl-containing
complexes. (iii) Substituting the chloride counterion for an
iodide anion in complexes 6a,c,e increases the hydrophobicity
of the complexes in comparison to their analogous chloride
complexes 4a,c,e. The higher lipophilicity of complexes 6a,c,e
was anticipated, as a larger iodide anion with a higher molar
mass provides greater polarizability than a chloride anion. The
in vitro cytotoxicity of silver-NHC complexes 4−6 was
determined using MTT-based assays following a 96 h drug-
exposure period. Compounds were initially tested for their
activity against pancreatic adenocarcinoma cell line Panc 10.05
and the noncancerous ARPE-19 cell line. Compounds with a
selectivity index (defined as the ratio of mean IC50 values for
ARPE-19 cells divided by the mean IC50 in cancer cells) higher
n
(p > 0.05) when the N substituent is methyl or butyl. The
reverse is observed when the N substituent is hydroxyethyl,
with chloride-containing complex 4e displaying greater
cytotoxicity in comparison to its iodide analogue 6e against
Panc 10.0 cells (p < 0.05). This difference was cell line
dependent, however, with 6e showing greater activity against
BE cells in comparison to 4e (although this did not reach
statistical significance; p > 0.05). These differences in trend
indicate that there is a fine balance between groups within the
overall complex, with modification of one moiety influencing
the effect of another.
All of the silver complexes were evaluated against the
noncancerous retinal epithelial cell line ARPE-19 to evaluate
Figure 4. Bar chart to show IC50 values for complexes 4−6 in comparison to those of cisplatin against Panc 10.05, MIA PaCa-2, and BE cancerous
cell lines and the ARPE-19 noncancerous cell line.
D
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Table 1. Responses of Panc 10.05, Mia-PaCa-2, and BE Cancerous Cell Lines and the ARPE-19 Noncancerous Cell Line to
a
Silver-NHC Complexes 4−6
cell line
Panc 10.05
Mia-PaCa-2
BE
ARPE-19
cisplatin
4a
1.7 0.4 (3.75)
7.1 3.7 (7.04 )
2.8 2.0 (2.26)
11.7 1.6 (4.27 )
0.7 0.3 (9.71)
18.0 4.4 (2.77 )
6.4 0.9
>50
b
b
b
4b
4c
4d
4e
5a
5b
5c
5d
5e
6a
9.4 5.6 (1.22)
13.5 2.8 (1.42)
5.8 1.4 (1.78)
1.9 0.7 (17.47)
16.1 0.9 (1.66)
16.1 1.9 (1.71)
16.8 1.8 (0.82)
13.8 1.8 (3.35)
6.5 4.5 (6.30)
10.0 6.8 (2.41)
3.8 1.9 (2.25)
20.5 12.6 (1.27)
6.3 4.8 (1.82)
6.7 1.1 (1.71)
11.5 2.9
19.2 3.7
10.2 0.8
32.5 6.6
26.6 7.8
27.5 7.5
13.8 6.5
46.3 4.7
41.2 6.2
24.2 2.6
8.7 1.5
26.0 1.7
7.0 1.6 (1.46)
4.9 1.8(6.62)
8.4 1.2 (1.21)
8.5 6.8 (3.80)
8.6 1.9 (4.78)
5.7 2.3 (4.23)
4.5 3.9 (1.91)
10.6 3.2 (2.28)
9.0 4.3 (4.56)
3.3 1.2 (7.31)
3.6 0.9 (2.42)
3.1 0.5 (8.51)
6c
6e
a
Values presented are IC50 (μM) SD for three independent experiments. The values in parentheses represent the selectivity ratio defined as the
b
IC50 value for ARPE-19 cells divided by the IC50 value for each of the cancer cell lines tested. Minimum possible value as the value for ARPE-19 is
50 μM (highest concentration tested).
Figure 5. RCVs recorded at 40 V s−1 of a DOPC-coated Pt/Hg electrode (black) in the presence of 50 μM silver(I)-NHC complex (left) or
clotrimazole-derived imidazolium salt (right) (red), recovery of DOPC (blue) in PBS at pH 7.4, and RCVs in the presence of 2 mM of cisplatin or
AgNO3 (red) in PBS at pH 7.4 (bottom).
their selectivity toward cancerous cells (Table 1). Complexes
4a,e and 5e show the greatest selectivity toward the cancerous
Panc 10.05 over the noncancerous ARPE-19, with selectivity
ratios higher than that of cisplatin. Complex 4e, the most
active of the 13 complexes, is also the most selective toward
Panc 10.05, exhibiting a selectivity ratio of 17.47, which is
almost 5-fold the selectivity of cisplatin. Complex 4e also
exhibits the highest selectivity ratio for cancerous MIA PaCa-2
over noncancerous ARPE-19, while 6e displays the highest
selectivity for cancerous BE over ARPE-19. Across the panel of
cell lines, selectivity indices were comparable for compounds
4b,d and 6c (Table 1). For compounds 4e and 6a,e, however,
selectivity ratios were cell line dependent, reflecting inherent
differences in cancer cell sensitivity to these compounds
(Table 1). While the SI values for 6a (7.31) and 6e (8.51)
against BE cells were less than the SI for cisplatin (9.71), these
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results suggest that caution should be exercised with regard to
selecting compounds on the basis of SI values obtained for one
cell line.
has some steric or related effect which inhibits compound
penetration of the DOPC monolayer. A full understanding of
the extent of the interaction, however, can only be obtained by
analyzing the LOD values, which also depend on the slope of
the calibration curves (see the Experimental Section). The
interaction of the silver complexes with DOPC is irreversible,
as the RCVs of the DOPC do not return to the original state
after electrolyte flushing. In general, the silver complex−DOPC
interaction appears to have little to do with their rather low log
P values and is more associated with their structure, where the
clotrimazole group clearly promotes lipid monolayer inter-
actions through its aromaticity.
Clotrimazole has been evaluated against MCF7 and MDA-
MB-231 breast cancer cell lines and its activity compared with
the response of nontumorigenic MCF10A cells.54 While
clotrimazole completely and preferentially inhibited the
proliferation of MCF10A cells, there was selective cell kill
(as determined by trypan blue exclusion, LDH release, and the
MTT assay) in MCF7 and MDA-MB-231 cells. As the cells
were immediately analyzed following drug exposure (24 h
duration), the results are not directly comparable to the results
of this study but do indicate that clotrimazole itself has some
inherent selectivity for cancer cells in vitro.
Model membrane experiments have been carried out
previously with an extensive series of compounds, including
tricyclic antidepressant pharmaceuticals,47 aromatic hydro-
carbons,62 flavonoids,63 ionic liquids,64 nanomaterials,65 and
biomembrane-active59 and -inactive66 peptides. Significantly
this study is the first one carried out on the interaction of
organometallic compounds with the phospholipid sensor
element, although metal ion interactions with the element
have already been investigated.67 The clear conclusion from
previous studies is that the electrochemical model membrane
platform is responsive to any species which is biomembrane-
active. Biomembrane activity indicates any interaction that
alters the structure and organization of a biomembrane and can
be, but not always is, related to lipophilicity.62 Generally the
interaction is due to an association with the phospholipid polar
groups and/or phospholipid hydrocarbon tails of the
phospholipid bilayer. RCVs of the imidazolium ligand
precursors tested demonstrate interactions with DOPC similar
to those observed for the silver complexes (Figure 5, right),
with the identical structure-based interaction of variations in N
substituents and the presence/absence of chloride on the
triphenyl group. A comparison of interactions of ligand
precursors with their corresponding silver complexes generally
shows a slight decrease in the interactions of the imidazolium
salts from the interactions with the silver complexes. However,
most interestingly, the interaction of the clotrimazole ligand
precursors, unlike that of the derived silver-NHC complexes,
tends to be reversible, where the RCVs of the DOPC return in
most cases almost to the original state. In the PBS electrolyte,
Cl− is the common ion where the ligand counterion is Cl−. In
the case where the ligand counterion is I−, it is unlikely that the
electrolyte Cl− replaces the very much more polarizable I−,
where there will be an element of ion pairing with the aromatic
ligand cation. The evidence shows that the nature of the
counterion appears to affect the interaction and reversibility of
the ligand cation’s interaction with the model membrane, as
shown by compounds 1e and 2e, respectively (Figure 5). This
difference in interaction would not be observed if the
counterion I− was replaced by Cl−. Interestingly, neither
cisplatin nor AgNO3 interacts with DOPC (Figure 5, bottom).
This supports the knowledge of the contribution of a copper
influx transporter in mediating the accumulation of cisplatin in
the cell.68 The fact that AgNO3 does not interact with the lipid
sensor element indicates that the silver-NHC interaction is a
function of its organically bound environment. We note that
this study was focused on the synthesis, cytotoxicity, and
putative anticancer activity of clotrimazole silver complexes.
The ligand precursor biomembrane activity was investigated
together with that of the silver complexes to obtain a better
understanding of what role the substituted Ag atom played in
the complex bioactivity.
To help assess the potential effect of silver complexes on the
biomembrane, a membrane-based sensing device was utilized
to test the putative biomembrane activity of the 13 complexes
4−6 and cisplatin.47,55−60 This device consists of an electrode-
supported monolayer of dioleoylphosphatidylcholine (DOPC),
which has been validated against DOPC bilayer vesicles
showing identical interactions with biomembrane-active
compounds. The DOPC monolayer is deposited on a
microfabricated Pt/Hg electrode that is connected to an
online high-throughput flow system and utilizes rapid cyclic
voltammetry (RCV) to display changes in capacitance current
peaks when a voltage scan of 40 V s−1 is applied.56 The DOPC
monolayer undergoes two potential-induced phase transitions
represented by two characteristic sharp capacitance current
peaks from −0.4 to −1.2 V (vs Ag/AgCl). The two sequential
reversible transitions correspond to the entrance of electrolyte
into the layer coincident with pore formation and the
reorganization of the layer to form a patchy bilayer,
respectively.58 The interaction of the complexes with the
layer can be observed by changes in the current peak
configurations representative of phospholipid monolayer
damage as well as increases in the baseline capacitance current
informing on how well the complexes and/or water penetrate
monolayer sensor element.60
Initially, screening of silver complexes 4e, 5d,e, and 6a,c,d
prepared at 50 μM solutions displayed differences in
interactions with DOPC (Figure 5, left), which could be
related to variations in ligand N substituents and the presence/
absence of Cl on the triphenyl group. A comparison of
interactions of the hydroxyethyl-containing complexes 4e, 5e,
and 6e with DOPC indicates that the chloride on the triphenyl
group increases the interaction with the DOPC sensor
element, as 5e shows less interaction than the chloride-
containing 4e and 6e. The effect of chloride as an aromatic
substituent in promoting interaction with the lipid has been
shown previously.53,61 Changing the N substituent from a
hydroxyethyl in 6e to a methyl in 6a or an n-butyl in 6c
decreases the interaction with the sensor element, which
indicates that improving the water solubility through addition
of a hydroxyethyl substituent may improve the kinetics of
interaction at the monolayer surface. Conversely, changing the
N substituent to a benzyl appears to increase the interaction
with the DOPC, even more than for hydroxyethyl, which is
shown by the difference between the interactions shown by the
RCVs of 5d,e. This is not unusual, as aromatic rings are
reported to promote phospholipid monolayer interactions.53,61
The decreased interaction of 6e in comparison to 4e with the
DOPC layer can be attributed to the larger iodide atom on 6e,
which, although it increases the hydrophobicity of the complex,
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The interaction of these complexes with the DOPC-coated
Hg follows the received wisdom in that the chloride on the
triphenyl group promotes an interaction. The rather surprising
observations are that the polar hydroxyethyl N substituent also
promotes an interaction and that the inserted Ag atom causes
an interaction with the lipid layer to become irreversible. Such
irreversibility has been seen before from interactions of a
DOPC sensor element with hydrophobic substituted benzene
compounds and with the more polar amine narcotics.62 The
observation of the effect of the interactions on the capacitance
current−potential curves is interesting. Both the ligand
precursors and silver-NHC complexes induce a depression of
the capacitance current peaks and a positive potential peak
shift on initial interaction. This is likely due to the positively
charged ligand precursors and the partially positive Ag atom
being located within the lipid polar group region.62 With no
increase in the capacitance current baseline, no penetration of
the apolar region is indicated. Where a stronger interaction is
noted (4e and 2d), a significant increase in the capacitance
current baseline is more indicative of a complete disruption of
the monolayer organization which, in the case of the ligand
precursor (2d), the interaction is partially reversible. Also
noted is the interaction of compounds containing iodides (6a,c
and 1a,c) with the DOPC sensor element causing a slight but
significant hump in the capacitance current baseline (see the
Supporting Information), which presumably is the effect of the
iodide moiety penetrating the monolayer.
showed improved selectivity toward Panc 10.05 over cisplatin.
4e and 5e exhibit “intermediate lipophilicity”, where the
complex contains both hydrophobic and hydrophilic groups,
which plays a role in both cytotoxicity and selectivity. The
hydrophilic part, which in these complexes is the hydroxyethyl
group, increases their water solubility and improves their
transfer at the biointerface, while the hydrophobic triphenyl
group promotes an interaction with the lipid bilayers of
membranes. While the chloride-containing groups 4 and 5
appear to be consistent in their cytotoxicity trends, iodide-
containing complexes 6a,c,e show different trends in
cytotoxicity across the different cancerous cell lines, indicating
that their activity is tumor type dependent. The introduction of
a silver atom to the clotrimazole-derived compounds renders
their interaction with an electrode-supported DOPC sensor
element irreversible. The heterocyclic N-substituted hydrox-
yethyl chain and a chloride atom on the triphenyl group
enhance the activity of the ligand precursors and the silver(I)-
NHC complexes with the DOPC sensor element. The silver
complex showing the strongest interaction with the biomem-
brane-like sensor element had an IC50 value very close to that
of cisplatin. These results help validate this electrochemical
platform as an early-stage screen, i.e. prior to cell studies, for
pharma candidates where the mode of action is structure
related and lipid membrane based.
EXPERIMENTAL SECTION
■
From the LOD values of the complexes on the DOPC
sensor element (Table 2), 4e has the lowest value of 0.001 μM
General Methods. Where stated, manipulations were performed
under an atmosphere of dry nitrogen by means of standard Schlenk
line techniques. Anhydrous solvents were prepared by passing the
solvent over activated alumina to remove water, copper catalyst to
remove oxygen, and molecular sieves to remove any remaining water,
Table 2. LODs Using a Phospholipid Sensing Device and
IC50 Values toward the Panc 10.05 Cancerous Cell Line
1
via the Dow−Grubbs solvent system. H and 13C{1H} NMR spectra
compound
LOD (μM)
IC50 SD (μM)
were recorded on either a Bruker DPX300 or a Bruker AV500
spectrometer. The values of chemical shifts are given in ppm and
values for coupling constants (J) in Hz. Mass spectra were collected
on a Bruker Daltonics (micro TOF) instrument operating in the
electrospray mode. Microanalyses were performed using a Carlo Erba
MOD 1106 Elemental Analyzer. X-ray diffraction data were collected
on either a Bruker Nonius X8 diffractometer fitted with an Apex II
detector with Mo Kα radiation (λ = 0.71073 Å) or an Agilent
SuperNova diffractometer fitted with an Atlas CCD detector with Mo
Kα radiation (λ = 0.71073 Å) or Cu Kα radiation (λ = 1.5418 Å).
Crystals were mounted under oil on glass or nylon fibers. Data sets
were corrected for absorption using a multiscan method, and the
structures were solved by direct methods using SHELXS-97 and
refined by full-matrix least squares on F2 using ShelXL-97.69,70
Molecular graphics for all structures were generated using POV-RAY
in the X-Seed program.
4a
4e
5d
5e
6a
6e
0.002
0.001
0.3
0.07
0.05
0.025
7.1 3.67
1.9 0.74
13.8 1.77
6.5 4.55
10.0 6.79
20.5 12.58
and 5d has the highest value of 0.3 μM. Interestingly, 4e also
exhibits a low IC50 value of 1.9 0.74 μM, showing a
cytotoxicity against Panc 10.05 superior to those of the other
complexes tested and comparable to the IC50 value of cisplatin
(1.7 0.41 μM). 5d, on the other hand, has a relatively high
IC50 value of 13.8
factors, in addition to lipid biomembrane damage, contribute
to the cytotoxicity of the silver complexes.
1.77 μM. The data imply that several
Synthesis of Imidazolium Iodide 1a. Clotrimazole (0.20 g, 0.58
mmol) and methyl iodide (0.72 mL, 11.60 mmol) were dissolved in
dichloromethane (3 mL) and heated to 45 °C in the microwave for
15 min with stirring. Diethyl ether was added to the resulting pale
yellow solution, which induced precipitation of the product as an off-
white solid. This was filtered, washed with diethyl ether, and dried in
CONCLUSIONS
■
Thirteen clotrimazole-based silver(I)-NHC complexes have
been synthesized and fully characterized. The antiproliferative
activities of the silver complexes were examined against
pancreatic adenocarcinoma cell line Panc 10.05 and non-
cancerous retinal epithelial cells ARPE-19 and compared to
that of cisplatin. Selected complexes were also examined
against pancreatic adenocarcinoma cell line MIA PaC-2 and
colorectal carcinoma cell line BE. While most of the complexes
were less potent than cisplatin against Panc 10.05, complex 4e
showed potency comparable to that of cisplatin in addition to
showing 5-fold greater selectivity than cisplatin to Panc 10.05
over the noncancerous ARPE-19. Complexes 5e and 4a also
1
vacuo. Yield: 0.21 g (75%). H NMR (CDCl3, 300 MHz): δ 9.10 (s,
1H, NCHN), 7.75 (s, 1H, NCH), 7.50−7.11 (m, 14H, aromatic),
6.95 (s, 1H, NCH), 4.25 (s, 3H, CH3). 13C{1H} NMR (CDCl3, 75
MHz): δ 138.0 (CH), 137.9 (C), 137.4 (C), 135.1 (C), 132.9 (CH),
131.7 (CH), 131.5 (CH), 130.1 (CH), 192.5 (CH), 129.2 (CH),
128.1 (CH), 124.1 (CH), 123.6 (CH), 79.5 (C), 38.8 (CH3). HRMS
(ESI+): calcd for C23H20ClN2 [M − I]+, 359.1310; found 359.1311.
Anal. Calcd for C23H26ClIN2·1/3H2O: C, 56.06; H, 4.23; N, 5.68.
Found: C, 56.00; H, 4.10; N, 5.70. Single crystals suitable for X-ray
diffraction analysis were grown by the vapor diffusion of diethyl ether
into a concentrated solution of the product in dichloromethane.
G
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Synthesis of Imidazolium Iodide 1c. Clotrimazole (0.20 g, 0.58
mmol) and butyl iodide (0.67 mL, 5.80 mmol) were dissolved in
dichloromethane (3 mL) and heated to 45 °C in the microwave for
15 min with stirring. Diethyl ether was added to the resulting pale
yellow solution, which induced precipitation of the product as a white
solid. This was filtered, washed with diethyl ether, and dried in vacuo.
Synthesis of Imidazolium Chloride 2c. 2-Chlorotrityl chloride
(0.50 g, 1.60 mmol) was dissolved in dichloromethane (5 mL), and 1-
butylimidazole (0.21 mL, 1.60 mmol) was added. The mixture was
transferred to an ampule and heated at 45 °C for 24 h in a closed
system. Excess diethyl ether was added to the resulting yellow solution
1
to obtain a white solid. Yield: 0.30 g (43%). H NMR (CDCl3, 300
1
Yield: 0.18 g (59%). H NMR (CDCl3, 300 MHz): δ 9.18 (s, 1H,
MHz): δ 9.60 (s, 1H, NCHN), 8.19 (s, 1H, NCH), 7.47−6.98 (m,
15H, aromatic and NCH), 4.63 (t, 2H, CH2), 1.84 (pent, 2H, CH2),
1.28 (sext, 2H, CH2), 0.86 (t, 3H, CH3). 13C{1H} NMR (CDCl3, 75
MHz): δ 138.5 (C), 138.2 (CH), 137.5 (C), 135.3 (C), 132.9 (CH),
131.8 (CH), 131.4 (CH), 129.8 (CH), 129.3 (CH), 129.0 (CH),
127.9 (CH), 123.6 (CH), 122.7 (CH), 79.1 (C), 50.5 (CH2), 32.5
(CH2), 19.4 (CH2), 13.6 (CH3). HRMS (ESI+): calcd for
C26H26ClN2 [M − Cl]+, 401.1779; found, 401.1764. Anal. Calcd
for C26H26Cl2N2: C, 71.39; H, 5.99; N, 6.90. Found: C, 71.10; H,
6.20; N, 6.60.
NCHN), 7.71(s, 1H, NCH), 7.51−6.91 (m, 14H, aromatic), 6.74 (s,
1H, NCH), 4.63 (t, J = 7.3 Hz, 2H, CH2), 1.86 (m, 2H, CH2), 1.37
(m, 2H, CH2), 0.93 (t, J = 7.3 Hz, 3H, CH3). 13C{1H} NMR (CDCl3,
75 MHz): δ 140.3 (C), 138.1 (C), 137.1 (CH), 135.5 (C), 132.1
(CH), 131.4 (CH), 130.0 (CH), 129.7 (CH), 129.0 (CH), 127.9
(CH), 127.0 (CH), 123.6 (CH), 121.5 (CH), 79.1 (C), 50.9 (CH2),
32.3 (CH2), 19.2 (CH2), 13.5 (CH3). HRMS (ESI+): calcd for
C26H26ClN2 [M − I]+, 401.1785; found, 401.1778. Anal. Calcd for
C26H24ClIN2·1/2H2O: C, 58.06; H, 5.06; N, 5.21. Found: C, 58.30;
H, 4.90; N, 5.30. Single crystals suitable for X-ray diffraction analysis
were grown by the vapor diffusion of diethyl ether into a concentrated
solution of the product in dichloromethane.
Synthesis of Imidazolium Iodide 1e. Clotrimazole (0.50 g, 1.45
mmol) and iodoethanol (0.23 mL, 2.9 mmol) were dissolved in
dichloromethane (3 mL) and heated at reflux under nitrogen for 48 h,
resulting in a brown solution. Addition of ethyl acetate precipitated a
yellow soli,d which was filtered and washed with ethyl acetate.
Trituration with acetone gave the product as a white solid. Yield: 0.38
g (47%). 1H NMR (CDCl3, 300 MHz): δ 9.04 (s, 1H, NCHN), 7.75
(s, 1H, NCH), 7.52−7.10 (m, 14H, aromatic), 7.02 (s, 1H, NCH),
4.64 (t, J = 4.9 Hz, 2H, CH2), 3.99 (t, J = 4.9 Hz, 2H, CH2), 2.82
(broad s, 1H, OH). 13C{1H} NMR (CDCl3, 75 MHz): δ 138.1 (C),
137.8 (CH), 137.4 (C), 135.3 (C), 133.0 (CH), 131.5 (CH), 131.4
(CH), 130.0 (CH), 129.5 (CH), 129.2 (CH), 128.1 (CH), 123.5
(CH), 123.4 (CH) 79.3 (C), 59.9 (CH2), 52.8 (CH2). HRMS (ESI
+): calcd for C24H22ClN2O [M − I]+, 389.1421; found, 389.1419.
Anal. Calcd for C24H22ClIN2O: C, 55.78; H, 4.29; N, 5.42. Found: C,
56.00; H, 4.40; N, 5.90. Single crystals suitable for X-ray diffraction
analysis were grown by the vapor diffusion of diethyl ether into a
concentrated solution of the product in methanol.
Synthesis of Imidazolium Chloride 2a. 2-Chlorotrityl chloride
(0.50 g, 1.60 mmol) and 1-methylimidazole (0.50 mL, 6.27 mmol)
were dissolved in acetonitrile (10 mL) and heated at reflux for 24 h.
The solution was cooled to room temperature, and the product was
precipitated as a white solid using diethyl ether. Yield: 0.53 g (85%).
1H NMR (CDCl3, 300 MHz): δ 9.10 (s, 1H, NCHN), 7.75 (s, 1H,
NCH), 7.50−7.11 (m, 14H, aromatic), 6.95 (s, 1H, NCH), 4.25 (s,
3H, CH3). 13C{1H} NMR (CDCl3, 75 MHz): δ 139.1 (CH), 138.4
(C), 137.6 (C), 135.4 (C), 133.0(CH), 131.7 (CH), 131.4 (CH),
130.0 (CH), 129.5 (CH), 129.1 (CH), 127.9 (CH), 123.7 (CH),
123.4 (CH), 79.3 (C), 37.8 (CH3). HRMS (ESI+): calcd for
C23H20ClN2 [M − Cl]+, 359.1310; found, 359.1311. Single crystals
suitable for X-ray diffraction analysis were grown by the vapor
diffusion of pentane into a concentrated solution of the product in
dichloromethane.
Synthesis of Imidazolium Chloride 2d. 2-Chlorotrityl chloride
(0.50 g, 1.60 mmol) and 1-benzylimidazole (0.25 mL, 1.93 mmol)
were dissolved in acetonitrile (10 mL) and heated at reflux for 24 h.
The solution was cooled to room temperature, and a yellow solid was
precipitated using diethyl ether. This was filtered and washed with
ethyl acetate and acetone to give a white solid, which was dried in
1
vacuo. Yield: 0.14 g (20%). H NMR (CDCl3, 300 MHz): δ 9.92 (s,
1H, NCHN), 7.49 (s, 1H, NCH), 7.44−7.02 (m, 19H, aromatic),
6.88 (s, 1H, NCH), 5.90 (s, 2H, CH2). 13C{1H} NMR (CDCl3, 75
MHz): δ 157.7 (C), 138.9 (CH), 138.5 (C), 137.5 (C), 135.4 (C),
133.7 (CH), 132.9 (CH), 131.9 (CH), 131.4 (CH), 129.9
(CH),129.4 (CH), 129.3 (CH), 129.1 (CH), 129.1 (CH), 127.9
(CH), 123.5 (CH), 122.1 (C), 79.3 (CH2), 54.1 (CH2). HRMS (ESI
+): calcd for C29H24ClN2 [M − Cl]+, 435.1623; found, 435.1630.
Single crystals suitable for X-ray diffraction analysis were grown by the
vapor diffusion of diethyl ether into a concentrated solution of the
product in dichloromethane.
Synthesis of Imidazolium Chloride 2e. 2-Chlorotrityl chloride
(1.0 g, 3.20 mmol) was dissolved in dichloromethane (5 mL) in an
ampule, to which 1-(2-hydroxyethyl)imidazole (0.30 mL, 3.20 mmol)
was added. The solution was heated to 45 °C for 24 h in a closed
system. Excess pentane was added to the yellow solution, yielding a
yellow oil, which was further recrystallized from methanol/pentane to
obtain a yellow solid. Yield: 1.32 g (97%). 1H NMR (d6-acetone, 300
MHz): δ 8.98 (s, 1H, NCHN), 7.68 (s, 1H, NCH), 6.91 (s, 1H,
NCH), 7.87−7.12 (m, 14H, aromatic), 4.41 (t, J = 12 Hz, 2H, CH2),
3.87 (t, J = 12 Hz, 2H, CH2). 1H NMR (d4-MeOD, 300 MHz): δ 9.00
(s, 1H, NCHN), 7.83 (s, 1H, NCH), 7.64−7.20 (m, 15H, aromatic
and NCH), 4.41 (t, 2H, CH2), 3.89 (t, 2H, CH2). 13C{1H} NMR (d6-
acetone, 125 MHz): δ 139.0 (C), 138.2 (CH), 135.4 (C), 133.2 (C),
132.1 (CH), 131.8 (CH), 130.4 (CH), 129.8 (CH), 129.3 (CH),
128.5 (CH), 127.8 (CH), 124.6 (CH), 123.8 (CH), 120.6 (C), 79.2
(C), 61.6 (CH2), 60.2 (CH2). HRMS (ESI+): calcd for C24H22ClN2O
[M − Cl]+, 389.1421; found, 389.1425. Anal. Calcd for
C24H22Cl2N2O·1/4H2O: C, 67.06; H, 5.28; N, 6.52. Found: C,
67.20; H, 5.25; N, 6.70. Single crystals suitable for X-ray diffraction
analysis were grown by the vapor diffusion of diethyl ether into a
concentrated solution of the product in methanol.
Synthesis of Imidazolium Chloride 2b. 2-Chlorotrityl chloride
(0.50 g, 1.60 mmol) and 1-allylimidazole (0.48 mL, 4.40 mmol) were
dissolved in acetonitrile (10 mL) and heated at reflux for 24 h. The
yellow solution was cooled to room temperature, and a yellow oil
formed on addition of diethyl ether. The solution was decanted from
the oil, which was washed with ethyl acetate and triturated with
acetone to give the product as a white solid that was dried in vacuo.
Synthesis of Imidazolium Chloride 3a. Trityl chloride (6.80 g,
24.40 mmol) and 1-methylimidazole (2 g, 24.40 mmol) were
dissolved in acetonitrile (20 mL) and heated at reflux for 24 h. The
solution was cooled, and a white solid was obtained with the addition
1
of excess diethyl ether. Yield: 6.1 g, (69%). H NMR (300 MHz,
1
Yield: 0.37 g (55%). H NMR (CDCl3, 300 MHz): δ 9.84 (s, 1H,
CDCl3): δ 9.50 (s, 1H, NCHN), 7.90 (s, 1H, NCH), 7.42−7.12 (m,
15H, aromatic), 6.97 (s, 1H, NCH), 4.29 (s, 3H, CH3). 13C{1H}
NMR (125 MHz, CDCl3): δ 147.0 (CH), 139.6 (CH), 138.0 (CH),
135.9 (CH), 129.7 (CH), 129.3 (CH), 129.0 (CH), 128.0 (CH),
127.9 (CH), 127.2 (CH), 124.1 (CH), 123.8 (CH), (CH), 122.1
(CH), 119.9 (CH), 79.5 (C), 36.2 (CH3). HRMS (ESI+): calcd for
C23H21N2 [M − Cl]+, 325.1699; found, 325.1701. Anal. Calcd for
C23H21ClN2.2H2O: C, 69.60; H, 6.35; N, 7.06. Found: C, 69.80; H,
6.30; N, 7.40.
NCHN), 7.71 (s, 1H, NCH), 7.51−7.06 (m, 14H, aromatic), 6.96 (s,
1H, NCH), 6.08−5.95 (m, 1H, CH), 5.41−5.35 (m, 4H,
NCH2CHCH2). 13C{1H} NMR (CDCl3, 75 MHz): δ 138.8 (CH),
138.4 (C), 137.5 (C), 135.4 (C), 132.9 (CH), 131.8 (CH), 131.5
(CH), 130.8 (CH), 130.0 (CH), 129.5 (CH),129.1 (CH), 127.9
(CH), 123.5 (CH), 122.2 (CH), 121.9 (CH2) 79.4 (C), 52.9 (CH2).
HRMS (ESI+): calcd for C25H22ClN2 [M − Cl]+, 385.1466; found,
385.1471. Single crystals suitable for X-ray diffraction analysis were
grown by the vapor diffusion of diethyl ether into a concentrated
solution of the product in dichloromethane.
Synthesis of Imidazolium Chloride 3b. Trityl chloride (5.15 g,
18.50 mmol) and 1-allyl-1H-imidazole (0.58 g, 1.49 mmol) were
H
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dissolved in dichloromethane (20 mL) and heated at reflux for 24 h.
Addition of excess diethyl ether gave a white solid that was dried in
vacuo. Yield: 3.03 g, (42%). H NMR (300 MHz, CDCl3): δ 9.62 (s,
filtrate and the solid dried in vacuo to give the product as a white
crystalline solid. Yield: 0.39 g (62%). 1H NMR (300 MHz, CDCl3): δ
7.44−7.42 (m, 2H, NCH and aromatic), 7.36−7.09 (m, 14H,
aromatic), 6.95 (m, 1H, NCH), 3.88 (s, 3H, CH3). 13C{1H} NMR
(75 MHz, CDCl3): δ 140.9 (C), 140.0 (C), 135.7 (C), 132.5 (CH),
131.5 (CH), 130.6 (CH), 130.5 (CH), 128.4 (CH), 128.1 (CH),
127.8 (CH), 123.6 (CH), 119.7 (CH), 77.7 (C), 40.2 (CH3). HRMS
(ESI+): calcd for C46H38AgCl2N4 [2M − AgCl2]+, 825.1521; found,
825.1520. Anal. Calcd for C23H19AgCl2N2·2/3CH2Cl2: C, 50.87; H,
3.67; N, 5.01. Found: C, 50.90; H, 4.10; N, 4.60.
1
1H, NCHN), 7.81 (s, 1H, NCH), 7.41−7.11 (m, 15H, aromatic),
6.98 (s, 1H, NCH), 6.10−5.97 (m, 1H, CH), 5.43−5.35 (m, 4H, N−
CH2CHCH2). 13C{1H} NMR (125 MHz, CDCl3): δ 139.6 (CH),
138.5 (C), 138.0 (CH), 130.6 (CH), 130.3 (CH), 129.6 (CH), 129.6
(CH), 129.1 (CH), 128.9 (CH), 128.6 (CH), 127.9 (CH), 127.6
(CH), 126.6 (CH), 126.4 (CH), 125.7 (CH), 123.8 (CH), 122.2
(CH2), 52.8 (CH2). HRMS (ESI+): calcd for C25H23N2 [M − Cl]+,
351.1856; found, 351.4187. Anal. Calcd for C25H23ClN2: C, 72.02; H,
6.37; N, 6.72. Found: C, 71.80; H, 6.30; N, 6.90.
Synthesis of Silver Complex 4b. Imidazolium salt 2b (0.30 g,
0.71 mmol) and silver oxide (0.17 g, 0.71 mmol) were dissolved in
dichloromethane (8 mL) and heated at reflux for 24 h in the dark.
The mixture was cooled and filtered over Celite, with the solid being
washed with dichloromethane. The solvent was removed from the
yellow filtrate and the solid dried in vacuo to give the product as a
Synthesis of Imidazolium Chloride 3c. Trityl chloride (1.35 g,
4.84 mmol) and 1-butylimidazole (0.60 g, 4.84 mmol) were dissolved
in dichloromethane (20 mL) and heated at reflux for 24 h. Excess
diethyl ether was added to the solution to obtain an off white solid. A
saturated solution of NaHCO3 (20 mL) was added to the solid and
the product was extracted into dichloromethane (3 × 20 mL).
Recrystallization from dichloromethane/diethyl ether yielded a white
1
white solid. Yield: 0.14 g, (38%). H NMR (300 MHz, CDCl3): δ
7.36−7.04 (m, 15H, aromatic and NCH), 6.88 (s, 1H, NCH), 5.87
(m, 1H, NCH2CHCH2), 5.24−5.12 (m, 2H, N−CH2CHCH2), 4.71
(d, 2H, N−CH2CHCH2). 13C{1H} NMR (125 MHz, CDCl3): δ
139.9 (C), 135.8 (C), 132.7 (CH), 132.6 (CH), 131.7 (CH), 130.7
(CH), 130.6 (C), 128.5 (CH), 128.2 (CH), 128.0 (CH), 123.9
(CH), 119.5 (CH), 118.5 (CH2), 77.6 (C), 55.6 (CH2). HRMS (ESI
+): calcd for C50H44AgCl2N4 [2M − AgCl2]+, 877.1834; found,
877.1830. Anal. Calcd for C25H21AgCl2N2·1/2H2O: C, 55.89; H,
4.13; N, 5.21. Found: C, 55.50; H, 4.20; N, 5.00.
1
solid which was dried in vacuo. Yield: 0.99 g (51%). H NMR (300
MHz, CDCl3): δ 9.46 (s, 1H, NCHN), 7.99 (s, 1H, NCH), 7.43−
7.03 (m, 15H, aromatic), 7.01 (s, 1H, NCH), 4.69 (t, 2H, CH2), 1.85
(q, 2H, CH2), 1.31 (sext, 2H, CH2), 0.97 (t, 3H, CH3). 13C{1H}
NMR (75 MHz, CDCl3): δ 139.6 (CH), 138.1 (C), 137.6 (CH),
136.3 (CH), 135.3 (CH), 133.5 (CH), 131.3 (CH), 130.4 (CH),
129.6 (CH), 129.3 (CH), 128.9 (CH), 127.7 (CH), 126.3 (CH),
125.7 (CH), 124.3 (Ar), 123.9, (CH), 50.5 (CH2), 32.3 (CH2), 19.5
(CH2), 13.5 (CH3). HRMS (ESI+): calcd for C26H27N2 [M − Cl]+,
367.2169; found, 367.2119. Anal. Calcd for C26H27ClN2·4/3H2O: C,
73.14; H, 7.00; N, 6.56. Found: C, 73.10; H, 6.90; N, 6.80.
Synthesis of Silver Complex 4c. Imidazolium salt 2c (0.02 g,
0.05 mmol) and silver oxide (0.006 g, 0.03 mmol) were dissolved in a
mixture of anhydrous methanol (3 mL) and dichloromethane (3 mL)
in a Schlenk flask. The mixture was stirred at room temperature for 24
h in the dark. The mixture was filtered over Celite and the solvent
removed from the filtrate in vacuo to give the product as a light yellow
Synthesis of Imidazolium Chloride 3d. Trityl chloride (0.50 g,
1.79 mmol) and 1-benzylimidazole (0.28 g, 1.79 mmol) were
dissolved in dichloromethane (5 mL). The solution was transferred
to an ampule and heated at 45 °C for 24 h in a closed system. Excess
diethyl ether was added to the solution to acquire an off-white solid,
which was filtered and dried in vacuo. A saturated solution of
NaHCO3 (20 mL) was added to the solid, and the product was
extracted into dichloromethane (3 × 20 mL). Excess diethyl ether was
added to the solution, furnishing a white solid, which was dried in
1
solid. Yield: 0.005 g (20%). H NMR (300 MHz, CDCl3): δ 7.61−
6.88 (m, 16H, aromatic and NCH), 4.15 (t, 2H, N−
CH2CH2CH2CH3), 1.83 (m, 2H, N−CH2CH2CH2CH3), 1.57 (m,
2H, NCH2CH2CH2CH3), 0.92 (m, 3H, NCH2CH2CH2CH3).
13C{1H} NMR (75 MHz, CDCl3): δ 141.2 (C), 139.6 (C), 138.4
(CH), 135.4 (C), 132.4 (CH), 131.4 (CH), 130.3 (CH), 129.7
(CH), 129.0 (CH), 128.4 (CH), 128.1 (CH), 127.8 (CH), 123.6
(CH), 118.2 (CH), 77.4 (C), 53.2 (CH2), 33.2 (CH2), 19.5 (CH2),
13.7 (CH3). HRMS (ESI+): calcd for C52H50AgCl2N4 [2M −
AgCl2]+, 909.2460; found, 909.2609. Anal. Calcd for
C130H125Ag4Cl9N10, i.e. 3:1 Ag(NHC)Cl:[Ag(NHC)2]Cl: C, 60.57;
H, 4.89; N, 5.43. Found: C, 60.80; H, 4.80; N, 5.10.
1
vacuo. Yield: 0.51 g (71%). H NMR (500 MHz, CDCl3): δ 9.82 (s,
1H, NCHN), 7.71 (s, 1H, NCH), 6.93 (s, 1H, NCH), 7.46−7.11 (m,
20H, aromatic), 5.94 (s, 2H, CH2). 13C{1H} NMR (75 MHz,
CDCl3): δ 138.2 (CH), 135.5 (C), 129.6 (CH), 129.5 (CH), 129.4
(CH), 129.3 (CH), 129.2 (CH), 128.9 (CH), 127.9 (CH), 127.2
(CH), 123.7 (CH), 122.0 (CH), 121.0 (CH), 52.93 (CH2). HRMS
(ESI+): calcd for C29H25N2 [M − Cl]+, 401.2012; found, 401.2016.
Anal. Calcd for C29H25ClN2·H2O: C, 76.55; H, 5.98; N, 6.16. Found:
C, 76.10; H, 5.90; N, 6.60.
Synthesis of Imidazolium Chloride 3e. Trityl chloride (0.50 g,
1.80 mmol) was dissolved in dichloromethane (5 mL) in an ampule,
to which 1-(2-hydroxyethyl)imidazole (0.17 mL, 1.80 mmol) was
added. The solution was heated to 45 °C for 72 h in a closed system.
A white solid precipitated out of the solution, which was filtered off
and recrystallized from methanol/pentane, giving a white solid that
was further dried in vacuo. Yield: 0.66 g (94%). 1H NMR (300 MHz,
d4-MeOD): δ 8.89 (s, 1H, NCHN), 8.24 (s, 1H, NCH), 7.79 (s, 1H,
NCH), 7.50−7.24 (m, 15H, aromatic), 4.37 (t, 2H, J = 10.5 Hz,
CH2), 4.24 (t, 2H, J = 10.5 Hz, CH2). 13C{1H} NMR (300 MHz, d4-
MeOD): δ 141.4 (C), 139.4 (C), 130.8 (CH), 130.3 (CH), 129.9
(CH), 125.5 (CH), 123.9 (CH), 60.9 (CH2), 53.5 (CH2). HRMS
(ESI+): calcd for C24H23N2O [M − Cl]+, 355.1810; found, 355.1813.
Anal. Calcd for C24H23ClN2O·4/3H2O: C, 69.47; H, 6.23; N, 9.00.
Found: C, 69.00; H, 6.00; N, 9.26. Single crystals suitable for X-ray
diffraction analysis were grown by the vapor diffusion of diethyl ether
into a concentrated solution of the product in methanol.
Synthesis of Silver Complex 4d. Imidazolium salt 2d (0.10 g,
0.21 mmol) and silver oxide (0.05 g, 0.21 mmol) were dissolved in
dichloromethane (8 mL) and heated at reflux for 24 h in the dark.
The mixture was filtered over Celite with the solid being washed with
dichloromethane, and the solvent was removed from the filtrate. The
solid was dried in vacuo to give the product as an off-white solid.
1
Yield: 0.06 g (50%). H NMR (300 MHz, CDCl3): δ 7.46−7.10 (m,
20H, aromatic and NCH), 6.87 (s, 1H, NCH), 5.35 (s, 2H, CH2).
13C{1H} NMR (75 MHz, CDCl3): δ 141.0 (C), 139.9 (C), 135.8
(C), 135.6 (C), 132.7 (CH), 131.7 (CH), 130.7 (CH), 129.2 (CH),
128.8 (CH), 128.6 (CH),128.2 (CH), 128.1 (CH), 127.8 (CH),
127.1 (CH), 78.0 (C), 57.2 (CH2). HRMS (ESI+): calcd for
C58H46AgCl2N4 [2M − AgCl2]+, 977.2147; found, 977.2170. Anal.
Calcd for C29H23AgCl2N2·2H2O: C, 56.70; H, 4.43; N, 4.56. Found:
C, 56.80; H, 4.60; N, 4.10.
Synthesis of Silver Complex 4e. Imidazolium salt 2e (0.2 g,
0.47 mmol) and silver oxide (0.065 g, 0.28 mmol) were dissolved in a
solvent mixture of anhydrous methanol (10 mL) and anhydrous
dichloromethane (5 mL) in a Schlenk flask with 4 Å activated
molecular sieves. The mixture was stirred at room temperature for 24
h in the dark. The mixture was filtered over Celite and the solvent
removed from the filtrate in vacuo to give the product as a white solid.
Synthesis of Silver Complex 4a. Imidazolium salt 2a (0.50 g,
1.27 mmol) and silver oxide (0.29 g, 1.27 mmol) were dissolved in
dichloromethane (10 mL) and heated at reflux for 24 h in the dark.
The mixture was cooled and filtered over Celite, with the solid being
washed with dichloromethane. The solvent was removed from the
1
Yield: 0.13 g (56%). H NMR (300 MHz, d4-MeOD): δ 7.52−6.99
(m, 16H, aromatic and NCH), 3.65−3.60 (m, 4H, CH2). 13C{1H}
NMR (75 MHz, d4-MeOD): δ 142.3 (C), 141.6 (C), 137.2 (C),
133.8 (CH), 132.5 (CH), 131.9 (CH), 131.6 (CH), 129.6 (CH),
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129.1 (CH), 128.1 (CH), 124.9 (CH), 120.6 (CH), 78.7 (C), 62.5
(CH2), 56.6 (CH2). HRMS (ESI+): calcd for C48H42AgCl2N4O2 [2M
− AgCl2]+, 885.1732; found, 885.1747. Anal. Calcd for
C48H42AgCl3N4O2: C, 62.59; H, 4.60; N, 6.08. Found: C, 63.20; H,
5.30; N, 5.90. Single crystals suitable for X-ray diffraction analysis
were grown by the vapor diffusion of diethyl ether into a concentrated
solution of the product in methanol.
calcd for C58H48AgN4 [2M − AgCl2]+, 909.2927; found, 909.2964.
Anal. Calcd for C232H192Ag5Cl5N16, i.e. 2:3 Ag(NHC)Cl:[Ag-
(NHC)2]Cl: C, 71.07; H, 4.94; N, 5.72. Found: C, 70.80; H, 5.30;
N, 5.60. Single crystals suitable for X-ray diffraction analysis were
grown by the vapor diffusion of diethyl ether into a concentrated
solution of the product in dichloromethane.
Synthesis of Silver Complex 5e. Imidazolium salt 3e (0.1 g,
0.26 mmol) and silver oxide (0.04 g, 0.15 mmol) were dissolved in a
solvent mixture of anhydrous methanol (10 mL) and anhydrous
dichloromethane (5 mL) in a Schlenk flask. The mixture was stirred at
room temperature for 24 h in the dark. The mixture was filtered over
Celite and the solvent removed from the filtrate in vacuo to give a
white solid. Yield: 0.018 g (14%). 1H NMR (300 MHz, d4-MeOD): δ
7.63 (broad s, 1H, NCH), 7.48−7.00 (m, 15H, aromatic), 6.86
(broad s, 1H, NCH), 4.12 (t, 2H, CH2), 3.81(t, 2H, CH2). 13C{1H}
NMR (75 MHz, d4-MeOD): δ 144.0 (C), 131.4 (CH), 129.4 (CH),
129.2 (CH), 124.5 (CH), 120.8 (CH), 78.6 (C), 62.4 (CH2), 56.3
(CH2). HRMS (ESI+): calcd for C48H44AgN4O2 [2M − AgCl2]+,
817.2512; found, 817.2529. Anal. Calcd for C144H132Ag5Cl5N12O6, i.e.
4:1 Ag(NHC)Cl:[Ag(NHC)2]Cl: C, 60.83; H, 4.68; N, 5.91. Found:
C, 60.80; H, 4.50; N, 5.80.
Synthesis of Silver Complex 6a. Imidazolium salt 1a (0.40 g,
0.84 mmol) and silver oxide (0.19 g, 0.84 mmol) were dissolved in
dichloromethane (40 mL) and heated at reflux for 24 h in the dark.
The solution was filtered over Celite and the solid washed with
dichloromethane. The solvent was removed from the filtrate in vacuo
to give the product as a white crystalline solid. Yield: 0.21 g (43%). 1H
NMR (300 MHz, CDCl3): δ 7.34−6.95 (m, 16H, aromatic), 3.88 (s,
3H, CH3). 13C{1H} NMR (75 MHz, d6-DMSO): δ 139.8 (C), 134.8
(C), 132.2 (C), 131.0 (CH), 130.2 (CH), 128.2 (CH), 128.1 (CH),
127.8 (CH), 123.2 (CH), 121.1 (CH), 118.5 (CH), 76.8 (CH), 40.1
(CH3). HRMS (ESI+): calcd for C46H38AgCl2N4 [2M − AgI2]+,
825.1516; found, 825.1532. Anal. Calcd for C23H19AgClIN2·3/
2CH2Cl2: C, 40.81; H, 3.08; N, 3.89. Found: C, 40.90; H, 3.20; N,
4.10.
Synthesis of Silver Complex 6c. Imidazolium salt 1c (0.15 g,
0.28 mmol) and silver oxide (0.07 g, 0.28 mmol) were dissolved in
dichloromethane (15 mL) and heated at reflux for 24 h in the dark.
The solution was filtered over Celite and the solid washed with
dichloromethane. The solvent was removed from the filtrate in vacuo
to give a yellow solid, which was recrystallized from dichloromethane/
pentane to give the product as a yellow solid. Yield: 0.08 g (47%). 1H
NMR (300 MHz, CDCl3): δ 7.45−7.09 (m, 15H, aromatic and
NCH), 6.96 (s, 1H, NCH), 4.15 (t, 2H, N−CH2CH2CH2CH3), 1.80
(dt, 2H, NCH2CH2CH2CH3), 1.32 (m, 2H, N−CH2CH2CH2CH3),
0.94 (dd, 3H, NCH2CH2CH2CH3). 13C{1H} NMR (75 MHz,
CDCl3): δ 141.1 (C), 140.0 (C), 135.8 (C), 131.6 (CH), 130.7
(CH), 130.6 (CH), 128.5 (CH), 128.2 (CH), 128.1 (CH), 123.6
(CH), 118.2 (CH), 77.8 (C), 53.6 (CH2), 33.5 (CH2) 19.7 (CH2),
13.8 (CH3). HRMS (ESI+): calcd for C52H50AgCl2N4 [2M − AgI2]+,
909.2460; found, 909.2609. Anal. Calcd for C26H25AgClIN2·2/
3C5H12: C, 51.52; H, 4.86; N, 4.10. Found: C, 51.90; H, 5.20; N,
4.00. Single crystals suitable for X-ray diffraction analysis were grown
by the vapor diffusion of pentane into a concentrated solution of the
product in dichloromethane.
Synthesis of Silver Complex 5a. Imidazolium salt 3a (0.50 g,
1.38 mmol) and silver oxide (0.32 g, 1.38 mmol) were dissolved in
dichloromethane (30 mL) and heated at reflux for 18 h in the dark.
The mixture was filtered over Celite and the solvent removed from
the filtrate in vacuo to give a brown solid. Recrystallization from
dichloromethane/diethyl ether gave the product as a white solid.
1
Yield: 0.4 g (62%). H NMR (300 MHz, CDCl3): δ 7.34−7.20 (m,
15H, aromatic), 7.04 (s, 1H, NCH), 6.98 (s, 1H, NCH), 3.84 (s, 3H,
CH3). 13C{1H} NMR (75 MHz, CDCl3): δ 140.5 (C), 129.6 (CH),
128.5 (CH), 128.2 (CH), 127.9 (CH), 122.0(HC), 119.8 (CH),
36.12 (CH3). HRMS (ESI+): calcd for C46H40AgN4 [2M − AgCl2]+,
757.2301; found, 757.2314. Anal. Calcd for C23H20AgClN2·2/3H2O:
C, 57.58; H, 4.48; N, 5.84. Found: C, 57.90; H, 4.60; N, 5.40. Single
crystals suitable for X-ray diffraction analysis were grown by the vapor
diffusion of diethyl ether into a concentrated solution of the product
in dichloromethane.
Synthesis of Silver Complex 5b. Imidazolium salt 3b (0.58 g,
1.49 mmol) and silver oxide (0.35 g, 1.49 mmol) were dissolved in
dichloromethane (30 mL) and heated at reflux for 18 h in the dark.
The mixture was filtered over Celite and the solvent removed from
the filtrate in vacuo to give a colorless oil. Recrystallization from
dichloromethane/diethyl ether gave the product as a white solid.
1
Yield: 0.41 g (55%). H NMR (300 MHz, CDCl3): δ 7.37−7.19 (m,
15H, aromatic), 7.07 (s, 1H, NCH), 6.95 (s, 1H, NCH), 6.00 (m, 1H,
N−CH2CHCH2), 5.24 (m, 2H, N−CH2CHCH2), 4.75 (d, 2H, N−
CH2CHCH2). 13C{1H} NMR (75 MHz, CDCl3): δ 142.1 (C), 132.6
(NCH), 130.6 (NCH), 129.6 (CH), 128.9 (CH), 128.6 (CH), 127.7
(CH), 119.5 (HC), 117.5 (CH2), 55.6 (CH2). HRMS (ESI+): calcd
for C50H44AgN4 [2M − AgCl2]+, 809.2614; found, 809.2616. Anal.
Calcd for C25H22AgClN2: C, 60.81; H, 4.49; N, 5.67. Found: C,
60.61; H, 4.50; N, 5.60. Single crystals suitable for X-ray diffraction
analysis were grown by the vapor diffusion of diethyl ether into a
concentrated solution of the product in dichloromethane.
Synthesis of Silver Complex 5c. Imidazolium salt 3c (0.62 g,
1.53 mmol) and silver oxide (0.36 g, 1.53 mmol) were dissolved in
dichloromethane (30 mL) and heated at reflux for 18 h in the dark.
The mixture was filtered over Celite and the solvent removed from
the filtrate in vacuo to give a colorless oil. Recrystallization from
dichloromethane/diethyl ether gave the product as a white solid.
1
Yield: 0.21 g (26%). H NMR (300 MHz, CDCl3): δ 7.37−7.18 (m,
15H, aromatic), 7.05 (s, 1H, NCH), 6.93 (s, 1H, NCH), 4.13 (t, 2H,
CH2), 1.80 (quin, 2H, CH2), 1.31 (sext, 2H, CH2), 0.93 (t, 2H,
CH2). 13C{1H} NMR (75 MHz, CDCl3): δ 147.0 (C), 137.2 (CH),
129.7 (CH), 129.4 (CH), 129.1 (CH), 128.0 (CH), 127.8 (CH),
124.0 (CH), 122.6 (CH), 79.7 (C) 50.6 (CH2), 32.4 (CH2), 19.6
(CH2), 13.6 (CH3). HRMS (ESI+): calcd for C52H52AgN4 [2M −
AgCl2]+, 841.3240; found, 841.3245. Anal. Calcd for C26H26AgClN2:
C, 61.25; H, 5.14; N, 5.49. Found: C, 61.60; H, 5.15; N, 5.40. Single
crystals suitable for X-ray diffraction analysis were grown by the vapor
diffusion of diethyl ether into a concentrated solution of the product
in methanol.
Synthesis of Silver Complex 6e. Imidazolium salt 1e (0.32 g,
0.63 mmol) and silver oxide (0.14 g, 0.63 mmol) were dissolved in
dichloromethane (30 mL) and heated at reflux for 24 h in the dark.
The solution was filtered over Celite and the solid washed with
dichloromethane. The solvent was removed from the filtrate in vacuo
Synthesis of Silver Complex 5d. Imidazolium chloride 3d
(0.086 g, 0.19 mmol) and silver oxide (0.027 g, 0.12 mmol) were
dissolved in a mixture of anhydrous methanol (5 mL) and
dichloromethane (5 mL) in a Schlenk flask with activated 4 Å
molecular sieves. The mixture was stirred at room temperature for 24
h in the dark. The mixture was filtered over Celite and the solvent
removed from the filtrate in vacuo to give a light yellow solid. Yield:
1
to give the product as a white solid. Yield: 0.12 g (31%). H NMR
(300 MHz, d4-MeOD): δ 7.00−6.55 (m, 16H, aromatic and NCH),
3.83 (m, 2H, N−CH2CH2OH), 3.45 (m, 2H, N−CH2CH2OH).
13C{1H} NMR (75 MHz, CDCl3): δ 140.9 (C), 140.2 (C), 135.8
(C), 132.5 (CH), 131.5 (CH), 130.7 (CH), 130.4 (CH), 128.4
(CH), 128.1 (CH), 127.8 (CH), 123.2 (CH) 120.1 (CH), 77.3 (C),
62.6 (CH2), 55.6 (CH2). HRMS (ESI+): calcd for C48H42AgCl2N4O2
[2M − I]+, 887.1883; found, 887.1864. Anal. Calcd for
C48H42AgCl2IN4O2·1/2CH2Cl2: C, 55.22; H, 4.11; N, 5.31. Found:
C, 55.20; H, 3.80; N, 5.00.
1
0.01 g (10%). H NMR (300 MHz, CDCl3): δ 7.45−6.88 (m, 22H,
aromatic and NCH), 4.54 (s, 2H, CH2). 13C{1H} NMR (125 MHz,
CDCl3): δ 186.6 (C), 184.1 (C), 142.3 (C), 135.8 (C), 130.1 (CH),
129.9 (CH), 129.0 (CH), 128.5 (CH), 128.3 (CH), 127.4 (CH),
124.3 (CH), 119.6 (CH), 77.4 (C), 65.9 (CH2). HRMS (ESI+):
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Cytotoxicity Studies. Cells were incubated in 96-well plates, at 2
× 103 cells per well in 200 μL of growth media (RPMI 1640
supplemented with 10% fetal calf serum, sodium pyruvate (1 mM),
and L-glutamine (2 mM). Cells were incubated for 24 h at 37 °C in an
atmosphere of 5% CO2 prior to drug exposure. Silver compounds and
cisplatin were dissolved in dimethyl sulfoxide at a concentration of 25
mM and diluted with medium to obtain drug solutions ranging from
50 to 0.049 μM. The final dimethyl sulfoxide concentration was 0.1%
(v/v), which is nontoxic to cells. Drug solutions were applied to cells
and the cells incubated for 96 h at 37 °C under an atmosphere of 5%
CO2. The solutions were removed from the wells, and fresh medium
was added to each well along with 20 μL of MTT (5 mg mL−1), and
the mixture incubated for 4 h at 37 °C under an atmosphere of 5%
CO2. The solutions were removed, and 150 μL of dimethyl sulfoxide
was added to each well to dissolve the purple formazan crystals. A
plate reader was used to measure the absorbance at 540 nm. Lanes
containing medium only and cells in medium only (no drug) were
used as blanks for the spectrophotometer and 100% cell survival,
respectively. Cell survival was determined as the absorbance of treated
cells divided by the absorbance of controls and expressed as a
percentage. The concentration required to kill 50% of cells (IC50) was
determined from plots of percent survival against drug concentration.
The results were expressed as the mean standard deviation for three
independent experiments. All compounds and cisplatin were initially
tested against Panc 10.05 (pancreatic cancer cell line) and ARPE-19
(noncancer retinal epithethial cell line). The selectivity index (SI) was
determined (defined as the mean IC50 for ARPE-19 cells divided by
the mean IC50 for Panc10.05 cells). Any compound with an SI value
greater than cisplatin was selected for further evaluation against two
other cancer cell lines (MiaPaCa-2 and BE). A selection of
compounds with SI values lower than that of cisplatin was also
tested in order to determine whether or not SI values against
MiaPaCa-2 and BE cell lines remained low or whether cell-dependent
variations were observed. Statistical analysis was conducted using a t
test with significance defined as p < 0.05 or p < 0.01.
DOPC monolayers undergo two pronounced phase transitions
characterized by two capacitance current peaks. These peaks
correspond to structural changes of, and the redistribution of charges
and polar groups on, the monolayer interface. All assays were carried
out in pH 7.4 PBS with continuous scanning from −0.4 to −1.2 V.
The compounds were tested for 400 s, after which PBS was flushed
through for 400 s to allow for any recovery of the DOPC layer initial
structure to take place. Both compound interaction and DOPC layer
depuration took place with the monolayer undergoing two disruptive
reversible phase transitions where each molecule of lipid is in contact
with the aqueous phase. Increasing the concentration of the
compounds in test media gave rise to increased responses following
compound interaction with the DOPC layer. This was shown as
enhanced peak suppression. The effect of compound dose on the
RCV plot emphasizes the effect of compound type on the nature of
and sensitivity to their interaction with the DOPC-coated electrode.
The degree of RCV scan recovery is related to the degree of DOPC
structure restoration, which in turn is related to the degree of
reversibility of the interaction.
The LOD was measured by plotting a calibration graph of
capacitance current peak height vs compound solution concentration
following interaction with the respective compound. Subsequently the
reproducibility of the capacitance current peak height from the RCVs
of the DOPC-coated Hg electrode in PBS was estimated by taking 10
replicate measurements. The 3 times standard deviation (SD) of this
capacitance peak height was calculated as a control. Accordingly the
concentration of the test compound, which has an equivalent
suppressive effect on the capacitance current peak height relating to
3 times the SD of the control, was read off the calibration curve to
estimate the detection limit values. The detection limits were
estimated and quoted as the minimum concentration of compound
in water to elicit a response and these detection limits are related to
compound molecular structure and the known biological effect of
each compound. The LOD is inversely proportional to the ability of
the compound to disrupt the layer structure and organization.
Hydrophobicity Measurements. Equal volumes of octanol and
NaCl-saturated water were stirred at room temperature for 24 h and
separated to give octanol-saturated water and water-saturated octanol.
Accurate amounts of the complexes were dissolved in the water-
saturated octanol (25 mL). A 3 mL portion of octanol-saturated water
was placed in a centrifuge tube, and 3 mL of water-saturated octanol
containing the complex was layered on top. The samples were shaken
for 4 h using a Vibrax machine at 500g min−1. The layers were
separated, and the water-saturated octanol layer was retained for
analysis using UV/vis spectroscopy. Using the maximum absorbance
for each complex, the average of six runs was calculated, and
rearrangement of individual calibration graphs gave the final [C]org
value. The [C]org and [C]aq values were used to determine the
partition coefficient log P.
Biomembrane Sensing Device. Full details of the device, the
apparatus used, and operation are fully described in a previous
publication.56 A microfabricated Pt/Hg electrode coated with DOPC
(Avanti Polar Lipids, Alabaster, AL, US, >99% purity) was contained
in a sealed flow cell. In the RCV experiments the fabricated
rectangular platinum electrode on the wafer was employed as a
counter electrode and a Ag/AgCl/3.5 M KCl reference electrode was
inserted into the flow cell. All potentials in this paper are quoted
versus this reference electrode. A constant flow of 0.1 M KCl, calcined
at 600 °C for 2 h and buffered at pH 7.4 with 0.01 M phosphate
(hereinafter referred to as phosphate-buffered saline or PBS) was
passed over the Pt/Hg electrode using a peristaltic pump. The
electrodes were connected to a potentiostat (PGSTAT 30 Autolab
potentiostat Ecochemie, Utrecht, The Netherlands) interfaced to a
Powerlab signal generator (AD Instruments Ltd.) and controlled by
Scope(c) software. Rapid cyclic voltammetry (RCV) scans were
obtained by applying a sawtooth waveform from −0.4 to −1.2 V with
a ramp rate of 40 V s−1 applied to the electrode surface. In the
absence of Faradaic reactions, the current on the RCV plot is directly
proportional to the capacitance of the surface and is displayed as a
function of voltage. In response to the applied voltage ramp, the
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.organomet.0c00069.
■
sı
X-ray crystallographic information, expanded RCVs, and
NMR spectra (PDF)
Accession Codes
CCDC 1949011−1949023 contain the supplementary crys-
tallographic data for this paper. These data can be obtained
free of charge via www.ccdc.cam.ac.uk/data_request/cif, or by
emailing data_request@ccdc.cam.ac.uk, or by contacting The
Cambridge Crystallographic Data Centre, 12 Union Road,
Cambridge CB2 1EZ, UK; fax: +44 1223 336033.
AUTHOR INFORMATION
Corresponding Authors
■
Roger M. Phillips − School of Applied Sciences, University of
Huddersfield, Huddersfield HD1 3DH, U.K.;
Email: R.M.Phillips@hud.ac.uk
Andrew Nelson − School of Chemistry, University of Leeds,
Leeds LS2 9JT, U.K.; Email: A.L.Nelson@leeds.ac.uk
Charlotte E. Willans − School of Chemistry, University of Leeds,
Leeds LS2 9JT, U.K.; orcid.org/0000-0003-0412-8821;
Email: c.e.willans@leeds.ac.uk
Authors
Heba A. Mohamed − School of Chemistry, University of Leeds,
Leeds LS2 9JT, U.K.
Samantha Shepherd − School of Applied Sciences, University of
Huddersfield, Huddersfield HD1 3DH, U.K.
K
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overview of N-heterocyclic carbenes. Nature 2014, 510 (7506), 485−
496.
(15) Praetorius, J. M.; Crudden, C. M. N-Heterocyclic Carbenes: From
Laboratory Curiosities to Efficient Synthetic Tools; The Royal Society of
Chemistry: 2011; p 77.
(16) Wang, M. H.; Scheidt, K. A. Cooperative Catalysis and
Activation with N-Heterocyclic Carbenes. Angew. Chem., Int. Ed.
2016, 55 (48), 14912−14922.
(17) Rajabi, F.; Thiel, W. R. An Efficient Palladium N-Heterocyclic
Carbene Catalyst Allowing the Suzuki−Miyaura Cross-Coupling of
Aryl Chlorides and Arylboronic Acids at Room Temperature in
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Nicola William − School of Chemistry, University of Leeds,
Leeds LS2 9JT, U.K.
Helen A. Blundell − School of Chemistry, University of Leeds,
Leeds LS2 9JT, U.K.
Madhurima Das − School of Chemistry, University of Leeds,
Leeds LS2 9JT, U.K.
Christopher M. Pask − School of Chemistry, University of
Leeds, Leeds LS2 9JT, U.K.
Benjamin R. M. Lake − School of Chemistry, University of
Leeds, Leeds LS2 9JT, U.K.; orcid.org/0000-0002-5775-
1230
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.organomet.0c00069
Author Contributions
All authors have given approval to the final version of the
manuscript. The authors declare no competing financial
interests.
Funding
The work was funded through the Universities of Leeds and
Huddersfield, NERC (UK) grant NE/M021378/1, and the EU
HORIZON 2020 HISENTS program grant agreement number
685817.
Notes
The authors declare no competing financial interest.
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